220D-F2 from Rubus ulmifolius Kills Streptococcuspneumoniae Planktonic Cells and Pneumococcal BiofilmsSharmila J. Talekar1, Sopio Chochua1, Katie Nelson2, Keith P. Klugman1, Cassandra L. Quave2,3,
Jorge E. Vidal1*
1Hubert Department of Global Health, Rollins School of Public Health, Atlanta, Georgia, United States of America, 2Center for the Study of Human Health, Emory College
of Arts and Sciences, Atlanta, Georgia, United States of America, 3Department of Dermatology, School of Medicine, Emory University, Atlanta, Georgia, United States of
America
Abstract
Streptococcus pneumoniae (pneumococcus) forms organized biofilms to persist in the human nasopharynx. This persistenceallows the pneumococcus to produce severe diseases such as pneumonia, otitis media, bacteremia and meningitis that killnearly a million children every year. While bacteremia and meningitis are mediated by planktonic pneumococci, biofilmstructures are present during pneumonia and otitis media. The global emergence of S. pneumoniae strains resistant to mostcommonly prescribed antibiotics warrants further discovery of alternative therapeutics. The present study assessed theantimicrobial potential of a plant extract, 220D-F2, rich in ellagic acid, and ellagic acid derivatives, against S. pneumoniaeplanktonic cells and biofilm structures. Our studies first demonstrate that, when inoculated together with planktoniccultures, 220D-F2 inhibited the formation of pneumococcal biofilms in a dose-dependent manner. As measured by bacterialcounts and a LIVE/DEAD bacterial viability assay, 100 and 200 mg/ml of 220D-F2 had significant bactericidal activity againstpneumococcal planktonic cultures as early as 3 h post-inoculation. Quantitative MIC’s, whether quantified by qPCR ordilution and plating, showed that 80 mg/ml of 220D-F2 completely eradicated overnight cultures of planktonicpneumococci, including antibiotic resistant strains. When preformed pneumococcal biofilms were challenged with 220D-F2, it significantly reduced the population of biofilms 3 h post-inoculation. Minimum biofilm inhibitory concentration(MBIC)50 was obtained incubating biofilms with 100 mg/ml of 220D-F2 for 3 h and 6 h of incubation. 220D-F2 alsosignificantly reduced the population of pneumococcal biofilms formed on human pharyngeal cells. Our results demonstratepotential therapeutic applications of 220D-F2 to both kill planktonic pneumococcal cells and disrupt pneumococcalbiofilms.
Citation: Talekar SJ, Chochua S, Nelson K, Klugman KP, Quave CL, et al. (2014) 220D-F2 from Rubus ulmifolius Kills Streptococcus pneumoniae Planktonic Cells andPneumococcal Biofilms. PLoS ONE 9(5): e97314. doi:10.1371/journal.pone.0097314
Editor: Gunnar F. Kaufmann, The Scripps Research Institute and Sorrento Therapeutics, Inc., United States of America
Received November 22, 2013; Accepted April 17, 2014; Published May 13, 2014
Copyright: � 2014 Talekar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
Streptococcus pneumoniae (pneumococcus) is an important human
pathogen associated with high morbidity and mortality [1]. The
global burden of pneumococcal infection is especially high in
children, immunocompromised patients and the elderly causing
severe illnesses such as pneumonia, otitis media, bacteremia and
meningitis [2–4]. The pneumococcus causes 15 million cases of
serious diseases [1,5] and 1.6 million deaths each year including
,1 million child deaths in those under 5 years of age [6]. In the
US, the pneumococcus is responsible for more than 6 million cases
of otitis media, ,500,000 cases of pneumonia, ,50,000 cases of
bacteremia, and ,3,000 cases of meningitis. Only in the elderly,
the annual cost of pneumococcal diseases, to the US government,
is nearly $5.5 billion [7].
Colonization of the human nasopharynx, which occurs in early
childhood, and persistence in this niche are prerequisites for
pneumococcal disease [4,8]. The pneumococcus persists for
months in the nasopharynx forming specialized structures referred
to as biofilms [9,10]. Biofilms are structured communities of
bacterial cells enclosed in a self-produced polymeric matrix
composed of polysaccharides, proteins and nucleic acids that is
adherent to inert or living surfaces [11]. In comparison to their
planktonic counterparts (108 CFU/ml), biofilm bacteria reach a
much higher density (1011 CFU/ml) which may impact the
pharmacodynamics of antibiotics [12]. Pneumococci embedded in
these biofilms can migrate to other anatomic sites to cause severe
biofilm-associated diseases such as pneumonia, and otitis media,
[13–15]. From the lungs of patients with pneumococcal pneumo-
nia or the ear cavity when causing otitis media, planktonic
pneumococci can disperse from the biofilm structure and invade
sterile sites such as the blood stream or brain, to cause lethal
bacteremia or meningitis, respectively [16–18].
It has been estimated that ,60% of bacterial infections and up
to 80% of chronic infections are mediated by organisms producing
biofilms [19–21]. Besides pneumococcal pneumonia and otitis
media, other infectious diseases in which biofilms have been
implicated include dental plaque, gingivitis, endocarditis, muscu-
loskeletal infections, and urinary tract infections [22], chronic
wounds [23], coating contact lenses [24]. Biofilm-related infections
lead to longer hospital stays, recurrent infections, and increased
fatalities in the most recalcitrant infections [19,25–30]. Moreover,
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biofilm-associated cells are between 102 to 103 fold less susceptible
to antibiotics than their planktonic counterparts [26,31–33].
With the emergence of multi-drug resistance clones there is
increase in antibiotic resistance in S. pneumoniae strains [34].
Whereas intrinsic mechanisms of antibiotic resistance of plank-
tonic pneumococci have been thoroughly studied [35,36], the
specific mechanism by which pneumococcal biofilms increase
antibiotic resistance is under active investigation. A study by
Garcia-Castillo et al (2007) demonstrates that biofilms formed by S.
pneumoniae strains, isolated from cystic fibrosis patients, had a
significantly higher resistance to penicillin, tetracycline and
rifampicin than did the same strains grown as planktonic
pneumococci [37]. Increased resistance of biofilm cells, in
comparison to planktonic cultures, have also been reported for
amoxicillin, erythromycin, clindamycin and levofloxacin [38], and
for cefazolin and vancomycin [39]. More recent in vivo studies by
Marks et al (2012), using a mouse model of persistence, showed
that pneumococcal biofilms formed on nasopharyngeal tissue were
more resistant to gentamicin and penicillin G than did pneumo-
cocci dispersed from the biofilm structure [40].
Natural products and related structures are increasingly
becoming essential sources of new pharmaceuticals, because of
the immense variety of functionally relevant secondary metabo-
lites. Our previous studies discovered that an extract from the
plant Rubus ulmifolius Schott., Rosaceae (Elmleaf blackberry)
showed antimicrobial activity (ranging from 50–200 mg/ml of
crude extract) against Staphylococcus aureus strains with no damage
to human mammalian cells [41]. Sophisticated studies using LC-
UV/MS/MS (liquid chromatography-ultraviolet absorption tan-
dem mass spectrometry) revealed that the extract, hereafter
referred as 220D-F2, contained ellagic acid (EA) and several
ellagic acid derivatives (EADs) or sapogenin-related compounds.
220D-F2 was able to limit formation of biofilms made by
methicillin-resistant S. aureus (MRSA) strains belonging to different
linages, (i.e. USA100, USA200, etc.). Tested strains included
community-acquired, global epidemic and MRSA strain USA300
[41]. MRSA strains bear resistance to all penicillins and other b–lactam antimicrobial drugs and produce severe human diseases
such as skin and soft tissue infections (SSTIs), endovascular
infections, pneumonia, septic arthritis, endocarditis, osteomyelitis,
foreign-body infections, and sepsis [41–43]. Our previous exper-
iments also demonstrate therapeutic potential of 220D-F2 since it
decreased staphylococcal biofilm counts formed on catheters
in vitro, and offered a more significant reduction of biofilm counts
when incubated along with antibiotics such as daptomycin or
clindamycin [41].
The aim of the present study was to assess the antibacterial
effect of 220D-F2 against planktonic pneumococci and pneumo-
coccal early and mature biofilms. We demonstrate here that
220D-F2 kills planktonic pneumococci and therefore limits the
formation of biofilm structures. 220D-F2 was also able to eradicate
early and mature biofilms and biofilms made on human
pharyngeal cells. Together, our studies highlight the potential
prophylactic and therapeutic applications of 220D-F2 against
pneumococcal diseases involving either planktonic cells (i.e.
bacteremia and meningitis) or biofilm-related diseases such as
otitis media and pneumonia.
Materials and Methods
Bacterial Strain and Culture ConditionsStrains used in this study were reference genome-sequenced S.
pneumoniae strain D39 [44] (GenBank accession # NC_008533),
TIGR4 [45] and SPJV01. D39 is a virulent encapsulated type 2
strain [46], TIGR4 is another invasive isolate that belongs to
vaccine serotype 4 and SPJV01 a D39-derivative expressing the
green fluorescent protein (GFP) [47]. S. pneumoniae strain GA58771
is resistant to amoxicillin, cefuroxime, clindamycin, erythromycin,
penicillin and tetracycline. Strains TN33388 [48] and 7828–04 are
resistant to chloramphenicol, erythromycin, and intermediate
resistant to linezolid [49]. S. pneumoniae strains were cultured on
Trypticase Soy Agar plates with 5% Sheep Blood (BAP), Muller-
Hinton (MH) broth, or Todd-Hewitt broth containing 0.5% (wt/
vol) of yeast extract (THY).
Preparation of Extract 220D-F2220D-F2 was prepared as previously described [41]. Briefly,
voucher specimens of the plant were deposited at the Emory
University Herbarium (GEO) and bulk samples of the roots were
dried, ground into a fine powder and extracted in 95% ethanol.
The plant material was removed via vacuum filtration and the
solvent removed through rotary evaporation and lyophilization.
The resulting extract was resuspended in water and subjected to
successive partitioning in hexanes, ethyl acetate, and butanol using
a separatory funnel. The butanol partition was dried down, and
then further separated using a gravity column with a methanol and
dichloromethane gradient (the active fraction, 220D-F2 being
collected at 40:60 MeOH:CH2Cl2).
220D-F2 Quality Control TestingAll batches of 220D-F2 were subjected to high-performance
liquid chromatography (HPLC) analysis for the purpose of batch
to batch quality control using an Agilent 1260 Infinity system with
quaternary gradient pumps, inline degasser, autosampler, column
heater, diode array detector and acquisition system (OpenLab
CDS ChemStation, Agilent Technologies, Santa Clara, CA,
USA). An Agilent Zorbax Eclipse XDB-C18 Analytical
4.66250 mm, 5 micron column was used at a temperature of
40uC. The extract was dissolved in 5% DMSO in H2O and a
20 mg injection was eluted at a flow rate of 1 ml/min using a
gradient of 2 solvent systems: (A) 0.1% formic acid in H2O; (B):
0.1% formic acid in ACN. The mobile phase was 98% A at time 0,
88% at 34 min. with a 16 min. hold, 75% at 70 min. 5% at
82 min. with a 16 min. hold, followed by a hold at 98% for
15 min. Chromatograms were compared with those of the original
samples to ensure the presence of major peaks prior to use in
bioassays [41]. Quality tested batches of 220D-F2 were suspended
in DMSO (stock of 20 mg/ml or 50 mg/ml), sterile filtered
(0.2 mm), and stored in sterile vials at 280uC prior to use in all
bioassays.
Preparation of Inocula to Challenge Planktonic Cells andfor Biofilm AssaysAn overnight BAP culture of S. pneumoniae strains was used to
prepare a cell suspension in THY broth to an optical density at
600 nm (OD600) of 0.05 and incubated at 37uC in 5% CO2
atmosphere until the culture reached an OD600 of 0.2 (early log
phase). An aliquot (,76105 CFU/ml) was inoculated in duplicate
into either an 8-well glass slide (Lab-Tek, Rochester, NY) or a
polystyrene 24-well microtiter plate (Costar, Corning, NY)
containing THY broth and incubated at 37uC with 5% CO2 for
the indicated time.
Antimicrobial Effects of 220D-F2 Against Planktonic CellsTo study the effect of 220D-F2 on planktonic cells, strains were
inoculated in THY broth, in 24-well polystyrene microtiter plates,
and immediately treated with either DMSO or two different
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concentrations of 220D-F2 (100 mg/ml and 200 mg/ml). Treated
bacteria were statically incubated at 37uC with 5% CO2
atmosphere for 3 h and CFU/ml of planktonic cells or planktonic
cells-derivative biofilms were obtained as follows: planktonic cells
were gently removed, serially diluted in phosphate buffered saline
(PBS, pH=7.4) and plated onto BAP to obtain CFU/ml. Once
supernatant-containing planktonic cells were completely removed,
biofilms were gently washed with phosphate buffered saline (PBS)
to eliminate unbound bacteria, 1 ml of sterile PBS was added and
then biofilms were scraped thoroughly, including well edges.
Biofilm suspensions were serially diluted in PBS and plated on
BAP to obtain biofilm viable counts (CFU/ml). Colonies were
counted using the Bantex 920A colony counter (American Bantex
Corporation, Burlingame, CA).
Evaluation of Viability of Planktonic Cells by aFluorescence-based MethodA LIVE/DEAD BacLight bacterial viability kit L7012 (Invitro-
gen-Molecular Probes) was used to further visualize the viability of
planktonic cells challenged with different concentrations of 220D-
F2. Fluorescent dyes included in the kit incorporate, or not, into
bacterial cells as a function of membrane integrity, and therefore
viability. Staining procedure and concentrations of dyes were
utilized as per manufacturer’s recommendations. Preparations
were observed and photographed utilizing an inverted Evos fl
microscope (Advanced Microscopy Group).
Antimicrobial Effects of 220D-F2 against PreformedPneumococcal BiofilmsTo study the antibacterial effect of 220D-F2 on early and
mature biofilms (preformed biofilms), strains were inoculated in
THY broth and incubated for 3 or 8 h. The culture medium was
then completely removed and biofilms were added with fresh
THY broth. These preformed biofilms were treated with DMSO
or 220D-F2 (100 mg/ml or 200 mg/ml) and incubated for an
additional 3, 6 or 12 h at 37uC with 5% CO2 atmosphere. Biofilm
counts were then obtained as described above. The minimum
biofilm-inhibiting concentration (MBIC) was calculated as the
concentration of the extract in which biofilms were eradicated (i.e.
killed and/or dispersed) to a level$90% for MBIC90 or$50% for
MBIC50 in comparison to control wells only treated with DMSO.
Fluorescence Images of Pneumococcal Biofilms Treatedwith 220D-F2To obtain fluorescence images of biofilms treated with the
extract 220D-F2, SPJV01 (expressing gfp under regulation of the
maltose promoter) [50] was inoculated in THY broth supple-
mented with 2% maltose and immediately treated with either
DMSO or different concentrations of 220D-F2. These cultures
were statically incubated at 37uC with 5% CO2 atmosphere for
8 h. Planktonic cells were then removed and biofilm cells were
washed with PBS and fixed with 4% paraformaldehyde overnight.
The cells were blocked with 2% Bovine Serum Albumin (BSA) for
1 h and stained for 1 h at room temperature in the dark with a
polyclonal anti-S. pneumoniae antibody coupled to fluorescein
isothiocyanate (FITC; ViroStat, Portland, ME). Preparations were
washed once with PBS, mounted with Vectashield mounting
medium (Vector Laboratories, Burlingame, CA) and analyzed
with a Zeiss LSM 510 confocal microscope. Confocal images were
analyzed with LSM Image Browser, version 4.02.121. In another
set of experiments, preformed biofilms (8 h) were treated as above
for 3, 6 or 12 h. Since GFP production is quenched 8–10 h post-
inoculation [47], biofilm preparations were stained with DAPI
(100 nM) [49,6-diamidino-2-phenylindole] for 5 min at room
temperature and images photographed using an inverted Evos fl
microscope (Advanced Microscopy Group).
Cell CulturesHuman-derived pharyngeal Detroit 562 cells (ATCC CCL-198)
were regularly cultured in Minimum Essential Medium (MEM
1X) (Gibco, Grand Island, NY) supplemented with non-heat
inactivated 10% fetal bovine serum (FBS) (Atlanta Biologicals,
Flowery Branch, GA), 1% nonessential amino acids (Sigma, St.
Louis, MO), 1% glutamine (Sigma, St. Louis, MO), penicillin
(100 U/ml) and streptomycin (100 mg/ml). Cells were normally
harvested with 0.25% trypsin (Gibco, Grand Island, NY),
resuspended in the cell culture medium, and incubated at 37uCin a 5% CO2 humidified atmosphere.
The Bioreactor (biBio) with Living Cultures on HumanNasopharyngeal CellsPreparation of cell cultures for experiments in the bioreactor
(hereafter refered as biBio) was done essentially as recently
described [51,52]. Human pharyngeal cells installed in the biBio
were inoculated with an aliquot containing,76105 CFU/ml of S.
pneumoniae D39 strain and immediately perfused with sterile MEM
medium, with a low flow rate (0.20 ml/min), supplemented with
non-heat inactivated 10% fetal bovine serum and buffered with
1% HEPES (10 mM) (Gibco, Grand Island, NY). Biofilms were
allowed to form for 8 h and treated with DMSO or different
concentrations of 220D-F2 (200, 400 or 800 mg/ml) for an
additional 12 h period. Higher doses of 220D-F2 were required to
kill pneumococci in the biBio. Biofilms were then collected in
sterile PBS. The supernatant coming off the apical side of the
biBio, and containing planktonic cells, was also collected after
treatment with 220D-F2. Both biofilms and planktonic cells were
serially diluted in PBS and plated onto BAP to obtain viable counts
(CFU/ml). Each concentration was tested in duplicate (technical
replicates) and the experiments were conducted on two different
days.
Quantitative Minimum Inhibitory Concentration (qMIC)A modified quantitative MIC (qMIC) approach, was utilized
because the physical aspect (i.e. color, texture, etc.) of the 220D-F2
interfered with classic MIC readings. qMIC was based on
recommendations of the Clinical and Laboratory Standards
Institute (CLSI) broth microdilution guidelines [53]. Briefly, an
overnight culture of the strains was utilized to prepare a suspension
in physiological saline which was adjusted to the 0.5 McFarland
turbidity standard. An aliquot (100 ml) of this bacterial suspensionwas added to 11 ml of THY broth, or Cation Adjusted Muller-
Hinton broth (CAMHB) with 2–5% lysed horse blood (LHB) as
per CLSI guidelines, and used it to serially dilute the extract 220D-
F2 in a 96-well plate (Costar, Corning, NY) to obtain a final
concentration of 20, 40, 80, 160, or 320 mg/ml. Negative control
(THY or CAMHB with LHB), experimental control (bacterial
suspension with DMSO) and positive control (bacterial suspension)
were included. Treated bacterial suspensions were incubated at
37uC with 5% CO2 atmosphere for THY or 35uC at ambient air
for CAMHB with LHB for 20 to 24 hours.
To quantify the number of viable pneumococcal cells post-
incubation with 220D-F2, we utilized a molecular approach by
using quantitative (q)PCR reactions as follows: DNA was extracted
from 100 ml of bacterial suspension treated with 100 ml of TEbuffer containing 0.04 g/ml of lysozyme and 75 U/ml of
mutanolysin, using the automated BioMerieux NucliSENS easy-
220D-F2 Kills Streptococcus Pneumoniae
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MAG 2.0.1 instrument (BioMerieux, Durham, NC). Extracted
DNA (1 ml) was utilized as template in qPCR reactions (25 ml)targeting the lytA gene [54]. These reactions contained 1X
Invitrogen Platinum Quantitative PCR SuperMix-UDG, 200 nm
of each primer (Forward-59-ACGCAATCTAGCAGAT-
GAAGCA–39 and Reverse-59-TCGTGCGTTTTAATTC-
CAGCT-39) and 200 nm of the probe (59-FAM –
GCCGAAAACGCTTGATACAGGGAG –39 –BHQ1), and mo-
lecular biology grade water. Duplicate reactions were run in a
CFX96 real-time system thermal cycler (Bio-Rad, Hercules, CA)
with the following cycling parameters, initial denaturation at 95uCfor 2 min, followed by 40 cycles of denaturation at 95uC for 15 sec
and annealing and extension at 60uC for 1 min. To obtain
molecular CFU/ml, purified genomic DNA from S. pneumoniae
reference strain TIGR4 was serially diluted to prepare standards
representing 2.146101, 4.296101, 4.296102, 4.296103,
4.296104, or 4.296105 genome copies. A standard curve was
constructed and CFU/ml (D39 [44] and TIGR4 [45] encode only
one copy of the lytA gene) were calculated using the Bio-Rad CFX
manager software. qMIC’s experiments were repeated three times
on different days. Efficiency of these qPCR studies was always
between ,95–99%.
Quantitative MIC by Dilution and PlatingReference strain D39 and multi-drug resistant strain GA 58771
were chosen for quantitative MIC studies by dilution and plating.
Strains were essentially prepared and treated with different
concentrations of 220D-F2 as indicated above. At the end of the
incubation period, bacteria were serially diluted and plated on
BAP to obtain viable counts (CFU/ml). Experiments included
technical replicates and were conducted at least three times.
Extraction of DNA from S. pneumoniae StrainsStrains were grown overnight on blood agar plates. Bacterial
suspensions were made with 200 ml of sterile PBS and DNA
extracted from washed pellets using the QIAamp Mini kit
(Qiagen), following the manufacturer’s instructions. Final elution
was done in 100 ml of sterile DNA grade water and DNA
preparations were stored at 280uC until used.
Statistical AnalysisThe statistical analyses were performed using the Microsoft
Excel software (Microsoft corporation). Differences between mean
values were calculated by Student’s t-test, with p value less than
0.05 considered statistically significant.
Results
220D-F2 Inhibits Formation of Pneumococcal BiofilmsWe and others have previously demonstrated that biofilms
made by invasive S. pneumoniae strain D39 are completely formed
between 8 to 10 h post-inoculation. We also demonstrate that
D39, and SPJV01 a D39 isogenic derivative, produces more
biofilm biomass than strains TIGR4 or strain R6 [47]. To begin
exploring the inhibitory effects that 220D-F2 would have on these
structures, we inoculated SPJV01 with increasing amounts of
220D-F2, and treated bacteria were incubated for 8 h. As this
extract was dissolved in DMSO, for the control wells included
along with strain D39 the same volume of DMSO (ml) was utilizedas for each of the tested concentrations of 220D-F2. Our results
demonstrate dose-dependent inhibition of formation of pneumo-
coccal biofilms treated with 220D-F2 but DMSO did not have an
apparent inhibitory effect on the formation of biofilms (not shown).
Confocal microscopy micrographs of biofilms formed, when
incubated with DMSO, show normal structure (i.e. dense zones
of bacterial aggregates) whereas those structures formed when in
the presence of 220D-F2 show a dose-dependent decrease of
attached bacteria (Fig. 1).
Figure 1. Inhibition of pneumococcal biofilms by 220D-F2. SPJV01 was inoculated in 96 well-plates containing THY and treated with DMSO orthe indicated concentration of 220D-F2. Treated cultures were incubated for 8 h at 37uC. Micrographs of fluorescent biofilms were obtained byconfocal microscopy. Scale bar shown at the bottom left panel is valid for all panels. Shown is a representative of five independent experiments.doi:10.1371/journal.pone.0097314.g001
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220D-F2 Inhibits Formation of Pneumococcal Biofilms byKilling Planktonic CellsSince the above experiment inoculated 220D-F2 and planktonic
cells at the same time, to investigate whether inhibition of biofilm
formation was due to the killing of planktonic cells, viability of
planktonic cultures was evaluated by dilution and plating. In
comparison to DMSO-treated control, planktonic cultures treated
with either 100 mg/ml or 200 mg/ml of 220D-F2 showed a
significant reduction of bacterial viability (Fig. 2A). Fluorescence
micrographs of planktonic pneumococci treated with 220D-F2
and stained with the LIVE/DEAD assay confirmed the presence
of pneumococci with disrupted membranes (Fig. 2C). As expected,
as there was a reduction in planktonic cells, our studies
demonstrate that biofilm counts were also reduced compared to
biofilm counts in DMSO-treated control wells (Fig. 2B). Together,
these results indicate that 220D-F2 kills planktonic cultures of S.
pneumoniae strain D39, as early as 3 h post-inoculation, thereby
reducing the population of pneumococci that forms the biofilm
structure.
Inhibitory Effect of Extract 220D-F2 against BiofilmsWe next evaluated the potential of 220D-F2 as an inhibitor
against early and mature pneumococcal biofilms made by strain
D39 [47].
Early biofilm structures were treated with increasing amounts of
the extract 220D-F2 and then incubated for either 3 or 6 h. We
also treated mature biofilm structures with 220D-F2 for an
additional 12 h period. Preformed biofilms produced 3 h post-
inoculation and then additionally treated with 220D-F2 for 3 h
showed a significant reduction of the biofilm biomass (Fig. 3A).
Similarly, preformed biofilms produced 3 h post-inoculation and
then treated for an additional 6 h showed significant reduction of
biofilms (Fig. 3B). Biofilm counts were similarly obtained whether
100 mg/ml or 200 mg/ml of 220D-F2 was utilized. The calculated
minimum biofilm inhibitory concentration (MBIC) revealed that
treatment of early biofilms for 3 or 6 h with 100 or 200 mg/ml of
220D-F2 obtained MBIC50. Mature biofilms were similarly
challenged with 100 and 200 mg/ml of 220D-F2. Consistent with
a dose-dependent effect, mature pneumococcal biofilms treated for
3 h with 100 mg/ml of 220D-F2 did not show a significant
decrease of biofilm cells (Fig. 4A) whereas treatment for 6 or 12 h
with the same amount induced a significant decrease of biofilm
Figure 2. Killing of planktonic pneumococci by 220D-F2. S. pneumoniae strain D39 was inoculated in 24 well-plates containing THY andtreated with DMSO or the indicated concentration of 220D-F2; treated cultures were incubated for 3 h at 37uC. Planktonic cells were removed (A) andthen biofilms were washed and removed (B). Both populations were diluted and plated onto BAP to obtain CFU/ml. Error bars represent the standarderror of the mean calculated using data from two independent experiments processed in duplicate. Statistical significance (p#0.05) was calculatedusing a non-parametric one-tailed Student’s t-test (*). C) Planktonic pneumococci treated for 3 h were also stained by the LIVE/DEAD assay andimaged using a fluorescent microscope. Panels show the merge of the green and red channel. White arrows points out dead pneumococci. Scale baris valid for all panels.doi:10.1371/journal.pone.0097314.g002
220D-F2 Kills Streptococcus Pneumoniae
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counts (Figs. 4B and 4C). The calculated MBIC of preformed
mature biofilms treated for 3, 6 or 12 h with 100 and 200 mg/ml
of 220D-F2 resulted in a MBIC50 or MBIC90, respectively.
Altogether, our results demonstrate that 220D-F2 induces a
significant reduction in preformed biofilm cells.
To further explore whether the 220D-F2-treated biofilm
structure had been dispersed, i.e. detached from the substrate,
biofilms were stained by fluorescence. Fluorescence micrographs
of mature biofilms treated for 3, 6 or 12 h with 50 mg/ml of 220D-
F2 revealed moderate detachment of the biofilm structure.
However, when incubated with 100 or 200 mg/ml for 12 h,
biofilms had been almost completely detached from the substrate
(Fig. 5).
220D-F2 Kills Pneumococcal Biofilms and Planktonic CellsFormed on Human Pharyngeal CellsTo assess whether 220D-F2 would kill S. pneumoniae growing in a
more natural environment, strain D39 was inoculated in the biBio
and incubated for 8 h. The biBio simulates the nasopharyngeal
environment as it has human pharyngeal cells and a continuous
flow of nutrients. S. pneumoniae in the biBio was then incubated with
different amounts of 220D-F2 and the viability of biofilms and
planktonic cells was evaluated by dilution and plating. Whereas
200 or 400 mg/ml did not change the biofilm biomass (Table 1),
incubating the biBio with 800 mg/ml of 220D-F2 for 12 h
significantly reduced pneumococcal biofilms (MBIC50) (Table 1).
At the same concentration counts of planktonic cells were also
significantly reduced (MBIC90).
Molecular Studies of Minimum Inhibitory Concentration(MIC)Our results indicate that 220D-F2 may have potential
therapeutic applications against pneumococcal diseases. There-
fore, we next investigated the minimum inhibitory concentration
of 220D-F2 utilizing a quantitative (q)MIC protocol. This
modified protocol included molecular quantification, and bacterial
counts, of S. pneumoniae D39 cells (CFU/ml) after exposure to those
tested 220D-F2 dilutions. Our molecular approach utilized a
quantitative PCR assay targeting the autolysin gene lytA [54].
Planktonic cells were diluted to the recommended cell density and
incubated with the vehicle (DMSO) or serial dilutions of 220D-F2
starting at 20 mg/ml. In comparison to the untreated control,
treatment with DMSO, 20 or 40 mg/ml of 220F-D2 resulted in a
similar bacterial density (Table 2) whether incubated in THY or
CAMHB with LHB. In contrast, treatment with 80 mg/ml
(Table 2) reduced bacterial viability by four orders of magnitude
whereas 160, or 320 mg/ml reduced pneumococcal cells down to
the lower limit of detection of our assay (,100 CFU/ml)
Figure 3. Killing of early pneumococcal biofilms by 220D-F2. S.pneumoniae D39 was incubated for 3 h at 37uC after which earlybiofilms were washed and added with fresh THY containing theindicated concentration of 220D-F2 or DMSO. Treated biofilms wereincubated for (A) 3 or (B) 6 h at 37uC and then washed, diluted andplated onto BAP to obtain CFU/ml. Error bars represent the standarderror of the mean calculated using data from two independentexperiments; each experiment was processed in duplicate. Statisticalsignificance (p#0.05) was calculated using a non-parametric one-tailedStudent’s t-test (*).doi:10.1371/journal.pone.0097314.g003
Figure 4. Killing of mature pneumococcal biofilms by 220D-F2. S. pneumoniae D39 was inoculated and incubated for 8 h at 37uC after whichmature biofilms were washed and added with fresh THY containing the indicated concentration of 220D-F2 or DMSO. Treated biofilms wereincubated for (A) 3, (B) 6, or (C) 12 h at 37uC and then washed, diluted and plated onto BAP to obtain CFU/ml. Error bars represent the standard errorof the mean calculated using data from two independent experiments; each experiment was processed in duplicate. Statistical significance (p#0.05)was calculated using a non-parametric one-tailed Student’s t-test (*).doi:10.1371/journal.pone.0097314.g004
220D-F2 Kills Streptococcus Pneumoniae
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indicating that 220D-F2 had killed almost, if not all, pneumococci.
Similar treatment of reference strain TIGR4 or three other
antibiotic resistant strains resulted in similar eradication of
pneumococci (Table 2). Dilution and plating of pneumococcal
strains treated with different concentrations of 220D-F2 showed
similar bacterial counts as those obtained using our molecular
approach (Table 3).
Discussion
To the best of our knowledge, this is the first report of an agent
with antibacterial activity for S. pneumoniae planktonic cells, and
pneumococcal biofilms, obtained from a botanical product. 220D-
F2 was able to kill preformed pneumococcal biofilms reducing the
population of biofilm cells 6 or 12 h post-treatment with 200 mg/ml, to ,10% or ,1%, respectively.
A clear dose response effect allowing killing and detachment of
preformed pneumococcal biofilms was observed. Complete
detachment of the biofilm structure from the substratum was
observed after 12 h of treatment with 200 mg/ml (Fig. 5), which
correlated well with bacterial counts showing a reduction of,99%
of the biofilm biomass (Fig. 4C). Moreover, using a biofilm model
with human pharyngeal cells, treatment of pneumococcal biofilms
with 800 mg/ml of 220D-F2 induced a significant reduction of the
biofilm biomass (Table 1). The observation that a higher amount
of 220D-F2 was required to kill S. pneumoniae biofilms in the biBio
with human pharyngeal cells (800 mg/ml) vs the static model
(200 mg/ml) might be due to a combination of different factors.
Figure 5. Micrographs of 220D-F2 incubated with mature pneumococcal biofilms. S. pneumoniae D39 was inoculated and incubated for8 h at 37uC after which biofilms were washed and added with fresh THY containing the indicated concentration of 220D-F2 or DMSO. These treatedmature biofilms were incubated for 3, 6 or 12 h and after washes, the biofilm structure was stained with DAPI (100 nM). Stained biofilms were imagedby fluorescence. Scale bar shown is also valid for all panels.doi:10.1371/journal.pone.0097314.g005
Table 1. Treatment of S. pneumoniae D39 grown in the biBio on human pharyngeal cells.
S. pneumoniae CFU/ml
Planktonic cells
DMSO 5.72610563.706105
220D-F2:
200 mg/ml 2.02610668.436105
400 mg/ml 5.23610662.576106
800 mg/ml* 1.10610469.806103
Biofilms
DMSO 3.54610761.536107
220D-F2:
200 mg/ml 2.63610867.246107
400 mg/ml 1.12610863.256107
800 mg/ml* 1.08610763.936106
6standard error
*p,0.05 in comparison to DMSO.doi:10.1371/journal.pone.0097314.t001
220D-F2 Kills Streptococcus Pneumoniae
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For example, the biBio is constantly perfused, at a fixed rate
(200 ml/min), with cell culture medium containing 220D-F2 that
might delay the exposure time of this compound’s active molecules
with pneumococci [3]. Unknown metabolites produced by living
cultures of human pharyngeal cells exposed to pneumococci may
also cause indirect inhibition of 220D-F2. There is also an
increased biofilm biomass when those pneumococcal biofilms are
grown on human pharyngeal cells that may account for the
increased concentration of 220D-F2 required to kill pneumococci
in this in vivo model [40,51].
Only a few other molecules have proven effective against
pneumococcal biofilms. A recent study by Domenech et al (2011)
found a ,80% reduction of pneumococcal biofilms when
preformed biofilms were incubated with the amidase LytA, which
is encoded and produced by the pneumococcus and other related
streptococci [55]. Dispersion of ,70% of pneumococcal biofilms
have been also achieved incubating with 5-azacytidine (5-aza), an
analog of the pyrimidine nucleoside cytidine [56]. A paper by
Trapetti et al (2009) showed that neuraminidase inhibitors DANA
(i.e., 2,3-didehydro-2-deoxy-N-acetylneuraminic acid), zanamivir,
Table 2. Bactericidal activity of 220D-F2 against S. pneumoniae strains.
Strain/Treatment CFU/ml
D39
Untreated 5.48610863.916106
DMSO 4.33610862.916107
220D-F2:
20 mg/ml 1.61610862.186107
40 mg/ml 3.41610865.916107
80 mg/ml 2.70610461.406104
160 mg/ml #10060.00
320 mg/ml #10060.00
TIGR4
Untreated 1.43610762.596106
220D-F2 (80 mg/ml) #10060.00
TN33388*
Untreated 9.58610768.556106
220D-F2 (80 mg/ml) #10060.00
7828-04*
Untreated 1.57610863.666107
220D-F2 (80 mg/ml) #10060.00
GA58771*
Untreated 8.73610761.136107
220D-F2 (80 mg/ml) #10060.00
6standard error,*antibiotic resistant strain.Experiments were repeated three times.doi:10.1371/journal.pone.0097314.t002
Table 3. Bactericidal activity of 220D-F2 against S. pneumoniae strains quantified by culture.
Strain/treatment CFU/mL
D39
Untreated 3.54610861.496108
DMSO 1.00610867.616107
220D-F2 (80 mg/ml) ,10060.00
GA58771*
Untreated 6.82610762.956107
DMSO 1.97610761.376107
220D-F2 (80 mg/ml) ,10060.00
6standard error,*antibiotic resistant strain.Experiments were repeated three times.doi:10.1371/journal.pone.0097314.t003
220D-F2 Kills Streptococcus Pneumoniae
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and oseltamivir inhibit the capacity of pneumococci to form sialic
acid-dependent biofilms [57]. Whereas activity of 5-aza against
other pathogens has not been investigated, LytA and neuramin-
idase inhibitors appear to be specific to pneumococcal biofilms.
Studies within this work added a botanical derivative extract to
this list. Additional advantages of 220D-F2 include that it is also
active against biofilms made by antibiotic resistant, MRSA strains,
and does not induce apparent damage to a variety of in vitro
cultured eukaryotic cells [i.e. human kidney proximal tubular
(HK-2) cells, rat kidney (NRK-52E) cells, mouse kidney proximal
tubular cells, and mouse hepatocytes (AML12)] [41].
Detachment of biofilms was mainly mediated by reduction of
bacterial viability as the LIVE/DEAD assay confirmed disrupted
membranes (Fig. 2C). Planktonic pneumococci were almost
completely eradicated by 220D-F2 showing a MIC of 80 mg/ml.
These strains included multidrug resistant pneumococci as well as
another reference strain TIGR4. Unlike other natural products
that have been tested against planktonic pneumococci and S.
aureus, [58–60] we demonstrate in this work that active molecules
within 220D-F2 bear potential to treat both biofilm-related
diseases and those pneumococcal diseases mediated by planktonic
organisms [i.e. pneumococci growing in liquid microenvironments
such as in blood or cerebrospinal fluid (CSF)].
220D-F2 contains a mixture of ellagic acid and ellagic acid
derivatives and other minor constituents that remain to be
determined [41]. Whereas some of 220D-F2 bactericidal proper-
ties may be attributed to ellagic acid and its glycosidic derivatives,
the other constituents may also play a significant role in killing
planktonic pneumococci and pneumococcal biofilms. Ongoing
studies are focused on the fractionation and isolation of individual
constituents found in 220D-F2 for structural elucidation and
activity testing. We hypothesize that MICs and MBICs of the
individual constituents will be lower than those observed in this
study for 220D-F2.
This work also introduced the use of molecular reactions, along
with MICs, to quantify bacterial density post antimicrobial
challenge. We took advantage of quantitative reactions that have
been used by our group and others to molecularly quantify
pneumococcal load and therefore have quantitative, rather than
qualitative, MICs. Bacterial counts obtained using these reactions
correlated with those CFU/ml obtained by dilution and plating.
Data presented in Table 2 and 3 showed a dramatic drop when
planktonic pneumococci were incubated with 80 mg/ml. There-
fore, ,105 CFU/ml of planktonic pneumococci, as inoculated per
CLSI guidelines, were eradicated with 80 mg/ml of 220D-F2. A
similar pneumococcal load has been reported present in lung tissue
of pneumonia patients ($104 CFU/g) or in their secretions ($
105 CFU/ml) [61]. These data support further fractionation of
220D-F2 and testing against the pneumococcus and other
pathogens.
The possibility that qPCR inhibitors contained in the extract
interfered with our reactions was refuted as another set of control
reactions was added, ,400 pg of pure S. pneumoniae DNA to tubes
containing pneumococci treated with 80 mg/ml of 220D-F2, and
reactions detected and quantified a similar amount of this DNA
control.
In conclusion, this work represents the first study to evaluate the
bactericidal activity of the extract 220D-F2 against both S.
pneumoniae planktonic cells and pneumococcal biofilms. The
antibacterial potential of individual constituents within this
composition for the treatment of pneumococcal disease produced
by multidrug resistant strains, such as chronic otitis media, is
promising and worth pursuing. As pneumococcal strains resistant
to several antibiotics are increasingly emerging, the discovery of
new alternatives such as those active molecules present in 220D-F2
may provide therapeutic alternatives for treating pneumococcal
diseases in the future.
Acknowledgments
The authors thank Magderie Klugman for her exceptional support in some
laboratory procedures. Also thanks to Dr. Fuminori Sakai and Dr. Joshua
Shak for the critical input during the course of this project and all members
of the Vidal, Klugman and Quave laboratories for suggestions and
discussion. The authors also thank Dr. Lesley McGee from the Centers for
Disease Control and Prevention (CDC) and Dr. David Stephens and Dr.
Scott Chancey from Emory University School of Medicine for providing S.
pneumoniae antibiotic resistant strains.
Author Contributions
Conceived and designed the experiments: SJT SC KPK CQ JEV.
Performed the experiments: SJT SC JEV. Analyzed the data: SJT CQ
JEV. Contributed reagents/materials/analysis tools: KN KPK. Wrote the
paper: SJT JEV.
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